GSM6729499: Intermediate, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Fresh mouse brains were collected in ice-cold PIPES solution. Precancerous stage whole-mount SVZ and adjacent CC were dissected under a stereomicroscope68. Cancer samples were visually inspected, and fluorescence signals were examined using a UV gun (Nightsea). Mouse brain tissue was dissociated into a single cell suspension described previously. Briefly, after activating papain (Worthington) for 10 minutes, the tissue sample was incubated in a solution containing papain (0.003 g per 100 mg tissue) and 10 mg/ml DNase I (Worthington) for 15 minutes at 37 °C. The suspension was centrifuges, and the resulting pellet was resuspended in ovomucoid (Worthington), followed by further dissociation by mechanical digestion. The cell suspension was layered onto a 22 % Percoll (Sigma) solution and centrifuged for 10 minutes at 590 x g at 4 °C. The cell pellet was resuspended in ice-cold Hanks balanced salt solution (HBSS) containing 1 % bovine serum albumen (BSA) and 0.1 % glucose, and DAPI was added. DAPI- tdTomato+ cells were then enriched by FACS, and live cells were concentrated by centrifugation. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics except for Tumor R1 sample with v2 protocol). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.